The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe 31P NMR. The broad, asymmetric lineshape of the 31P NMR spectrum of isolated brush-border vesicles demonstrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18 mM), demonstrating that the vesicles are highly stable, corresponding to their biological function. However, the emergence of a narrow peak superimposed on the broad lineshape indicates that a small proportion of the membrane phospholipids has reached isotropic motion, which may correspond to external or internal micellar structures. Incubation with mixed micelles of fatty acids and taurochlorate show that long-chain fatty acids enhance the membrane-perturbing effect of taurocholate while short-chain, water-soluble fatty acids do not, suggesting a difference in the absorption mechanisms.